The effect of hydrostatic pressure as a stressor in connection with increased stress tolerance and shock proteins has been studied in chondrocytes, yeast and bacteria, but not yet in gametes and embryos.
The physiological mechanisms by which microorganisms adapt to sublethal stresses are not yet understood well. Recent studies describe that instabilities caused by sublethal cold shock in the normal protein synthesis in bacteria are overcome by the synthesis of so-called cold-shock proteins (CSPs) (Phadtare et al., 1999). These CSPs are suspected to have many functions such as RNA chaperones (Graumann and Marahiel, 1999) or transcription activators (LaTena et al., 1991); it is assumed that they also play a role in the protection against freezing (Wouters et al., 1999). Further investigations found that the production of CSPs is induced not only by cold shock, but also by other environmental stresses. In Escherichia coli, for example, a type of CSP is produced by nutritional stress (Yamanaka et al., 1998).
Another trial showed that high hydrostatic pressure treatment provoked the production of certain cold-induced proteins and heat shock proteins (Welch et al., 1993). Since both cold shock and high pressure treatment increase CSP levels, trials were conducted about the possibility of cross-protection. Wemekamp-Kamphuis et al. (2002) found that the level of survival after pressurization of cold-shocked Listeria monocytogenes was 100-fold higher than that of the cells growing at 37° C.
Hydrostatic pressure in the range of 30-50 MPa usually inhibits the growth of various organisms: the initiation of DNA replication is one of the most pressure-sensitive intracellular processes (Abe et al., 1999). The effects vary in severity depending upon the magnitude and duration of compression (Murakami and Zimmerman, 1973). The cell membrane is noted as a primary site of pressure damage (Palou et al., 1997). High hydrostatic pressure treatment can alter the membrane functionality such as active transport or passive permeability and therefore may perturb the physico-chemical balance of the cell (Yager and Chang, 1983; Aldridge and Bruner, 1985; Macdonald, 1987; Schuster and Sleytr, 2002; Routray et al., 2002). The application of pressure can lead to changes in protein structure, including partially or completely unfolded conformations. Pressure can cause the denaturation of proteins (Schmid et al., 1975; Weber and Drickamer, 1983; Jaenicke, 1991; Gross and Jaenicke, 1994; Silva et al., 2001). Recent reports state that hydrostatic pressure enhances the production of shock proteins (Welch et al., 1993; Wemekamp-Kamphuis et al., 2002).
The physical or biochemical processes at altered pressure conditions are governed by the principle of Le Chatelier: all reactions that are accompanied by a volume decrease speed up considerably (Murakami and Zimmerman, 1973; Welch et al., 1993; Palou et al., 1997). The accumulation of the pressure effects is lethal beyond a certain level: while irreversible changes of some biomolecules take place at higher pressures, at 300 MPa most bacteria and multicellular organisms die. Though tardigrades—in their active state they die between 100 and 200 MPa—can survive up to 600 MPa if they are in a dehydrated state (Seki and Toyoshima, 1998). An early publication showed that biological systems are able to tolerate high pressures as long as the pressure is reduced slowly (Johnson et al., 1954). Pribenszky et al. (2003, 2004) also explored the possibility of gradual retrieval of the pressurized embryos and found that gradual release of pressure significantly improves survival.
In response to various stress stimuli, heat shock genes are induced to express heat shock proteins (HSPs). Previous studies have revealed that expression of heat shock genes is regulated both at transcriptional and posttranscriptional level, and the rapid transcriptional induction of heat shock genes involves activation of the specific transcription factor, heat shock factor 1 (HSF1). Furthermore, the transcriptional induction can vary in intensity and kinetics in a signal- and cell-type-dependent manner. Kaarniranta et al. (1998) demonstrated that mechanical loading in the form of hydrostatic pressure increases heat shock gene expression in human chondrocyte-like cells. The response to continuous HHP was characterized by elevated mRNA and protein levels of HSP70, without activation of HSF1 and transcriptional induction of hsp70 gene. The increased expression of HSP70 was mediated through stabilization of hsp70 mRNA molecules. Interestingly, in contrast to static pressurization, cyclic hydrostatic loading did not result in the induction of heat shock genes. The findings of Kaarniranta et al. (1998) showed that hsp70 gene expression is regulated post transcriptionally without transcriptional induction in chondrocyte-like cells upon exposure to high continuous hydrostatic pressure. They suggested that the posttranscriptional regulation in the form of hsp70 mRNA stabilization provides an additional mode of heat shock gene regulation that is likely to be of significant importance in certain forms of stress.
Previously, the present inventors found that a sublethal shock, high hydrostatic pressure (HHP), significantly improves the post-thaw survival of frozen mouse blastocysts (Pribenszky et al., 2005a, WO2005022996). Similarly, at semen cryopreservation, the average post-thaw motility was significantly superior with pressure pre-treatment in each of the pressurized bovine semen compared to the samples frozen without previous pressurization. The result clearly describes the beneficial effect of a previous pressure treatment to the post thaw motility of cryopreserved bull semen (Pribenszky et al., 2005b). Further investigations for exploring the biological background and biochemical change during the HHP process will unveil the mechanism of its protective effects. These studies, however, involve the cryopreservation of the biological material after the HHP pre-treatment, which is clearly not possible, or of low efficiency with a variety of biological material.
The process of semen chilling or storing at temperatures above 0° C. is well established to store spermatozoa for a short period of time [Hackett, et al., 1982; Pinto, 1999; O'Shea et al., 1964]. With optimal semen treatment (dilution) and storage at optimal temperatures the semen can be inseminated with acceptable fertility results (but with obviously reduced conception rates compared to fresh semen insemination) within 1-2 days post collection [Gill et al., 1970; Goodman and, Cain, 1993; Harrop, 1954; Ijaz and Ducharme, 1995; Katila et al., 1997] These methods follow very similar basic steps:    1. Semen collection.    2. Semen dilution at body temperature.    3. Optional centrifugation of the diluted semen. Re-extending the semen to adjust the optimal sperm-concentration.    4. Keeping the (re)extended semen at room temperature or 4-5° C. or any temperature that is above the freezing point of the sample.    5. Insemination of the semen.
Similarly to spermatozoa that suffer a loss of viability during storage, the survival capacity of embryos or oocytes also reduce once removed from their physiological maternal surrounding (for example for in vitro culture, activation, embryo transfer, splitting, sex determination, biopsy, in vitro maturation, ICSI, cloning or any type of biotechnological procedure). For this reason improving the viability/survival capacity of gametes and embryos before or after any procedure including from routine storage, insemination or transfer as far as the most complex biotechnological procedure is of great scientific and economic importance.
Similarly, for example during preservation of microorganisms, such as bacteria (e.g. freeze-drying), the viability of microorganisms is greatly compromised. Improving the efficacy of any process that comes together with improved viability bears immense scientific and economic significance.
As it is clear from the above, there is still a need in the art for the improvement of the viability of biological material that is widely used in biotechnology protocols.
The present inventors surprisingly found that by applying a hydrostatic pressure challenge the viability of biological materials can be improved significantly, and by the application of the method, many state of the art biotechnology protocols can be accomplished more efficiently. The present specification shows a wide range of examples on this finding: after applying the present method to embryo transfer or insemination, the conception rate and birth rate improved; by applying the present method to oocytes, their stress tolerance greatly increased, which resulted in improved cleavage rate and higher blastocyst formation rate; by applying the present method to semen, and then by following state of the art dilution and storage, the motility of the spermatozoa was preserved for a significantly longer period of time.
It was also surprising that the improvements were substantial even when avoiding to apply temperatures below the freezing point of the medium during any stage of the storage and/or manipulation of the biological material. This finding has significant practical implications for the usability of the present and similar HHP methods.
In this context we must emphasize that the present inventive concept equally applies to any different biotechnical/biotechnological protocol or procedure used in the assisted reproductive technologies (ART) and other procedures, and the choice of those is not limited with respect to the invention. The only necessary step to include in the improved protocols is the step of hydrostatic pressure challenge; the parameters of which can be easily optimized by a person skilled in the art when following the teachings of the present description.
Because semen freezing yields poor post-thaw survival of spermatozoa at boars (and horses as well), the most common tool of breeding at these species is the insemination of fresh, extended, extended and cooled or extended and chilled semen. By the use of HHP pre-treatment semen is significantly better preserved at the given temperature, and also, the time of storage with higher quality is considerably increased. Similarly, in vitro and in vivo embryo production, in vitro culture of embryos, sexing, splitting, gene transfer, embryo transfer, oocyte maturation, activation, ICSI, cloning or any biotechnical/biotechnological procedure in the embryo, oocyte or sperm greatly reduce their viability/survival capacity. As an extrapolation of the above features, by the use of HHP pre-treatment gametes and embryos will enter any type of assisted reproductive technology (ART) or biotechnical/biotechnological procedure with an increased survival capacity.